This proposal describes a five-year training program for development of a career as a physician-scientist in neonatology and medical genetics, with particular expertise in early endoderm and lung development. The candidate is completing a unique five-year fellowship program that will lead to board certification in both neonatal-perinata medicine and medical genetics. This fellowship included three years of intensive research funded by the Ruth L. Kirschstein National Research Service Award (T32). During this time, Dr. Swarr has become particularly interested in the role that the non-coding genome, and lncRNAs in particular, plays in directing normal development, and when disrupted, how these non-coding elements contribute to human disease. During the proposed study period, Dr. Swarr will continue to develop his knowledge of endoderm and lung biology, acquiring additional skills in mouse genetics, epigenetics, stem cell biology, and bioinformatics through a combination of intensive laboratory work, graduate-level courses, conferences, and one-on-one mentoring. This project will be carried out under the mentorship of Edward E. Morrisey, PhD, a recognized leader in the field of pulmonary biology. Dr. Morrisey is a Professor of both Medicine and Cell and Molecular Biology, as well as the Scientific Director of the Institute for Regenerative Medicine at the University of Pennsylvania. He has mentored numerous postdoctoral fellows and graduate students. An Advisory Committee, consisting of Drs. Michael Beers, Klaus Kaestner, Ken Zaret, and Rebecca Simmons, will assist Dr. Morrisey in mentoring Dr. Swarr to independence. Each of these faculty members is a highly regarded scientist in his or her own right, and all have successfully mentored many graduate students, post-doctoral fellows, and trainee physician- scientists. The proposed research focuses on the role that long non-coding RNAs (lncRNAs) play during early endoderm and lung development. Although a number of critical transcription factors and molecular pathways have been identified that direct fundamental aspects of lung development, the molecular mechanisms by which these master regulatory signals are carefully coordinated remains poorly defined. Long non-coding RNAs (lncRNAs) have recently been recognized to provide a crucial layer of control over proper gene expression, playing essential roles in both normal development and human disease. Dr. Swarr, with others in the Morrisey laboratory, have recently demonstrated that lncRNAs are spatially correlated with transcription factors and regulate lung development (Herriges MJ and Swarr DT, et al. Genes Dev. 2014 Jun 15;28(12):1363-79). In this same study, Dr. Swarr identified a lncRNA located 2kb downstream of Foxa2, herein referred to as Falcor (for Foxa2-Adjaent Long non-COding RNA). The pioneering transcription factor Foxa2 is known to play important roles in early endoderm specification and subsequent differentiation of the lung. The central hypothesis of this proposal is that Falcor plays a critical role in regulating the proper expression of Foxa2, and as a result is an essential orchestrator of endoderm and lung development. In Aim 1, CRISPR-Cas9 technology will be employed to study the role of Falcor in both mouse and human development. In Aim 1a, a Falcor knockout mouse model, which has already been generated, will be used to study the effects of loss of Falcor in vivo. In Aim 1b, the role of Falcr in human development will be studied by differentiating Falcor human embryonic stem (ES) cell knockout lines into definitive endoderm, and ultimately, mature airway epithelium. In Aim 2, the molecular mechanisms by which Falcor regulates the expression of Foxa2 will be defined. A comprehensive proteomics-based approach will be used to identify proteins interacting with Falcor, and the functional significance of these interactions will be interrogated using a series o shRNA knockdown experiments. The research outlined in this proposal will provide novel insights into the function of lncRNAs in early endoderm and lung development, with potential impact for a broad range of lung diseases, from congenital malformations to adult lung cancer. Moreover, by completing this work, I will acquire the necessary technical skills and scientific expertise to become a physician-scientist in neonatal-perinatal medicine and medical genetics with expertise in the mechanisms by which the non-coding genome and epigenetic processes direct normal development, and how disruptions of these developmental processes lead to congenital malformations and pulmonary disease.